The use of supports in chromatographic systems is well known, as is the use of such systems for the chromatography of biological compounds, such as proteins, nucleic acids, viruses, and dextrans.
Heretofore, it has become common to use an inorganic support in chromatographic systems since such supports commonly provide good mechanical stability. Inorganic supports commonly utilized are, for example, glass, silica or alumina. With the advent of high speed chromatography, supports providing good mechanical stability have become increasingly important since such supports must withstand the relatively high pressures encountered in this type of chromatography. Inorganic supports, however, while providing good mechanical stability, often adversely affect at least some biological compounds and hence are not suitable for use with such compounds. By way of example, inorganic supports such as silica or glass beads denature or adsorb many enzymes. Obviously, while inorganic supports have proved useful due to the mechanical stability provided, the use of chromatographic systems including such supports has been seriously limited due to the adverse effect of such supports upon many biological compounds.
Carbohydrates have also been used extensively as supports and stationary phases in chromatographic systems. An early use was in the paper chromatographic separation of amino acids. In this type of separation, the cellulose of paper adsorbs the more polar components of the solvent system and acts as a stationary phase support for liquid-liquid partition separations.
Later it became known that carboxymethyl and diethylaminoethyl derivatives of cellulose could be used in the ion exchange purification of compounds. In this type of separation, water causes the cellulose to swell into a matrix into which protein may diffuse. Ion exchanging groups covalently linked in this matrix then partition molecules on the basis of their charge.
Still later, it became known that polysaccharides could be used in the preparation of steric exclusion and affinity chromatography columns. The ability of carbohydrate to imbibe water and the ease with which derivatization reactions could be carried out in polysaccharide matrices were again important factors in their selection.
Finally, gas chromatography columns have been prepared by coating solid supports with monosaccharides such as mannitol and sorbitol. At the present time, carbohydrates or carbohydrate derivatives have been used successfully in the preparation of gas, liquid-liquid partition, ion exchange, steric exclusion, and affinity chromatography columns.
The success of carbohydrates as chromatographic supports is based primarily upon: the ability of carbohydrates to imbibe large quantities of polar solvents which act as a stationary phase and cause the carbohydrate to swell into a hydrophilic matrix; the chemical stability of the formed hydrophilic matrix and the ease with which it is derivatized with ion exchange groups, ligands, and specific functional groups; and the ability of carbohydrates to stabilize sensitive biological compounds such as proteins.
The use of carbohydrates as a support, however, did not prove to be satisfactory for all uses since the carbohydrate support was found to have low mechanical stability. Where attempts were made to achieve more rapid analysis by increasing the flow rate of carbohydrate columns, the resulting higher pressures caused the bed to collapse. Thus, the primary objection to current carbohydrate supports is that analysis times are too long, and hence such supports are not suitable for high speed chromatographic systems.
For high speed chromatographic systems, the currently accepted technique for decreasing analysis time is to pump liquid through columns. Modern high speed liquid chromatography columns packed with silica or glass (inorganic) supports are capable of withstanding pressures of 300 to 400 atmospheres and flow rates 50 to 100 times greater than carbohydrate columns. But as brought out hereinabove, while silica and glass particles have good mechanical stability, they lack many of the desirable characteristics of carbohydrates such as, for example, the ability of carbohydrates to handle proteins without adsorbing or denaturing the same.
Heretofore, however, no support has been suggested or utilized that combines both good mechanical stability and superior separation characteristics in handling compounds such as biological compounds to provide a chromatographic system that offers quality of performance with minimum time requirements.